Co-biosynthesis Of 3-Hydroxypropionic Acid And 1,3-Propanediol By Lactobacillus Reuteri

3-Hydroxypropionic acid(3-HP)and 1,3-propanediol(1,3-PD)are two high value-added platform compounds,which are widely used in food,pharmaceutical,chemical and other industries.Biosynthesis of 3-HP and 1,3-PD has gained tremendous attention due to its economic competitiveness,environmental friendliness,and less byproducts.At present,research on co-biosynthesis of 3-HP and 1,3-PD by Lactobacillus reuteri during whole cell biotransformation of glycerol has been reported extensively,however,general and major challenges of which are the low titer of target products and accumulation of toxic intermediate metabolite 3-hydroxypropionaldehyde(3-HPA).Therefore,screening robust strains and developing novel whole-cell catalytic systems with appropriate transformation configurations could be expected to provide a potential alternative for large-scale exploitation of 3-HP and 1,3-PD.Considering these facts,the following work was carried out:(1)A potent L.reuteri with high yield of 3-HP plus 1,3-PD was isolated and identified from infant feces by enrichment culture,colorimetric screening and High Performance Liquid Chromatography(HPLC),which was further designated as L.reuteri FXZ014.(2)The factors involved mostly in the first stage were optimized by the one-factor at-a-time technique.The optimal conditions were as follows: glycerol supplementation40 m M,culture time 12 h and anaerobic condition.Then the optimal conditions of the second stage were obtained by response surface methodology(RSM),which were biomass 13 g/L CDW,glycerol concentration 330 m M and biotransformation time 3 h.Eventually,biotransformation of 3-HP and 1,3-PD was conducted under these conditions,getting a final net titer of 18.68 g/L and net productivity of 6.23 g/L/h.(3)The gab D4 and pdu Q gene coding succinate-semialdehyde dehydrogenase(Gab D4)and 1,3-propanediol dehydrogenase(Pdu Q)was cloned and expressed in Escherichia coli BL21 respectively.The generated engineering strains were named as E.coli Gab D4 and E.coli Pdu Q accordingly.Then,the Gab D4 and Pdu Q co-expression vector was constructed and introduced to E.coli BL21,obtaining the recombinant E.coli Gab D4-Pdu Q.Thereafter,six co-culture systems(CS)were designed using various combinations and biomass ratio of L.reuteri FXZ014 and engineered E.coli strains(E.coli Gab D4,E.coli Pdu Q and E.coli Gab D4-Pdu Q)constructed above,among of which,the co-culture system-2(CS-2)showed highest titers of 3-HP and 1,3-PD.The CS-2was composed of 50% L.reuteri FXZ014 and 50% E.coli Gab D4-Pdu Q.The biotransformation process of CS-2 was further optimized.The optimal conditions were:biomass 20 g/L,glycerol concentration 60 g/L and the bioconversion time 4 h.Under these conditions,the net titer and productivity of CS-2 was 45.15 g/L and 11.28 g/L/h,compared with the the mono-culture of L.reuteri FXZ014,the increase of which was141.7% and 81%,respectively.(4)The expression level of Gab D4 and Pdu Q in engineered E.coli were fine-tuned by untranslated region(UTR)engineering,which resulted five UTR engineering strains.Subsequently,the CS-2 was modified by replacing the E.coli Gab D4-Pdu Q with the UTR engineering strains,generating five novel co-culture systems.As a result,the CS-2-V3 accumulated minimal 3-HPA and provided the highest net titer of 3-HP and 1,3-PD,indicating that the key pathways of CS-2-V3 were well rebalanced.The net titer of3-HP and 1,3-PD was 85.63 g/L,which was improved by 89.6% from those produced by CS-2.In this context,modified technological configurations were developed and applied to CS-2-V3 for increasing the glycerol consumption,as well as the production of 3-HP and 1,3-PD.In the two-step biotransformation approach,the 3-HP and 1,3-PD net titer increased to 119.3 g/L.In the mode of fed-batch by sequentially feeding glycerol,the net titer of these two products further improved,which reached 159.92g/L.Furthermore,the technology of fed-batch by sequentially feeding both glycerol and fresh cells was investigated,resulting a dramatic improvement of 3-HP and 1,3-PD titer of 125.9 and 88.46 g/L,respectively.The final net production and productivity of3-HP plus 1,3-PD were 214.39 g/L and 8.93 g/L/h.Compared with the mono-culture of L.reuteri FXZ014,the net production of 3-HP and 1,3-PD increased by 10.4-fold,while the productivity improved by 43.3%.